日本原子力研究開発機構機関リポジトリ - JAEAand UMP are known to be dominant in aqueous solution.9 All six species (both neutral and (de)protonated CMP, dTMP, and

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Title Nitrogen K-edge X-ray absorption near edge structure of pyrimidine-containing nucleotides in aqueous solution

Author(s) Hiroyuki Shimada, Hirotake Minami, Naoto Okuizumi, Ichiro Sakuma, Masatoshi Ukai, Kentaro Fujii, Akinari Yokoya, Yoshihiro Fukuda, and Yuji Saitoh

Citation Journal of Chemical Physics, 142(17), 175102 (2015)

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Nitrogen K-edge x-ray absorption near edge structure of pyrimidine-containingnucleotides in aqueous solutionHiroyuki Shimada, Hirotake Minami, Naoto Okuizumi, Ichiro Sakuma, Masatoshi Ukai, Kentaro Fujii, AkinariYokoya, Yoshihiro Fukuda, and Yuji Saitoh Citation: The Journal of Chemical Physics 142, 175102 (2015); doi: 10.1063/1.4919744 View online: http://dx.doi.org/10.1063/1.4919744 View Table of Contents: http://scitation.aip.org/content/aip/journal/jcp/142/17?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides inaqueous solution J. Chem. Phys. 141, 055102 (2014); 10.1063/1.4891480 C–C bond unsaturation degree in monosubstituted ferrocenes for molecular electronics investigated by acombined near-edge x-ray absorption fine structure, x-ray photoemission spectroscopy, and densityfunctional theory approach J. Chem. Phys. 136, 134308 (2012); 10.1063/1.3698283 Communication: Near edge x-ray absorption fine structure spectroscopy of aqueous adenosine triphosphateat the carbon and nitrogen K-edges J. Chem. Phys. 133, 101103 (2010); 10.1063/1.3478548 Pyrimidine and halogenated pyrimidines near edge x-ray absorption fine structure spectra at C and N K-edges: experiment and theory J. Chem. Phys. 133, 034302 (2010); 10.1063/1.3442489 Electronic structure of ZnO nanorods studied by angle-dependent x-ray absorption spectroscopy andscanning photoelectron microscopy Appl. Phys. Lett. 84, 3462 (2004); 10.1063/1.1737075

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THE JOURNAL OF CHEMICAL PHYSICS 142, 175102 (2015)

Nitrogen K-edge x-ray absorption near edge structureof pyrimidine-containing nucleotides in aqueous solution

Hiroyuki Shimada,1,a) Hirotake Minami,1 Naoto Okuizumi,1 Ichiro Sakuma,1Masatoshi Ukai,1 Kentaro Fujii,2 Akinari Yokoya,2 Yoshihiro Fukuda,3 and Yuji Saitoh31Department of Applied Physics, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo184-8588, Japan2Advanced Science Research Center, Japan Atomic Energy Agency, Tokai-mura, Naka-gun, Ibaraki 319-1195,Japan3Synchrotron Radiation Research Center, Japan Atomic Energy Agency, Sayo-gun, Hyogo 679-5148, Japan

(Received 10 March 2015; accepted 17 April 2015; published online 7 May 2015)

X-ray absorption near edge structure (XANES) was measured at energies around the N K-edgeof the pyrimidine-containing nucleotides, cytidine 5′-monophosphate (CMP), 2′-deoxythymidine5′-monophosphate (dTMP), and uridine 5′-monophosphate (UMP), in aqueous solutions and in driedfilms under various pH conditions. The features of resonant excitations below the N K-edge in theXANES spectra for CMP, dTMP, and UMP changed depending on the pH of the solutions. Thespectral change thus observed is systematically explained by the chemical shift of the core-levels ofN atoms in the nucleobase moieties caused by structural changes due to protonation or deprotonationat different proton concentrations. This interpretation is supported by the results of theoretical calcula-tions using density functional theory for the corresponding nucleobases in the neutral and protonatedor deprotonated forms. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4919744]

INTRODUCTION

DNA lesions are induced by ionizing radiation and cancause serious biological effects such as cell death and muta-tions. DNA lesions can be induced indirectly, by diffusiblereactive oxygen species such as OH· radicals produced fromirradiated water molecules surrounding the DNA molecule(indirect effect),1 or can be induced by direct energy transferfrom ionizing radiation to the DNA molecule (direct effect).Both pathways are estimated to be almost equally important.2

The physicochemical mechanism of the direct effect has beenstudied extensively.3 However, the mechanism by which theearliest step in the process occurs, namely, the primary forma-tion of base lesions, is not yet well understood, in part becausethe DNA molecule is in an aqueous environment. For example,Yokoya et al. pointed out the importance of hydrating watermolecules in the induction of base lesions by the direct effect.4

Clearly, a detailed investigation of the response of DNA toradiation in an aqueous environment is required.

Monochromatized soft X-rays from synchrotron radiationsources have been used to study the direct effect of radia-tion and to specify the primary atomic sites (C, N, and O)using inner shell excitation.5 N atoms are present in onlythe nucleobases and not in the DNA backbone; therefore, werecently examined the selective excitation of the nucleobasemoiety of nucleotides in aqueous solution by measuring theX-ray absorption spectra at energies near the N K-edge.6–8

Furthermore, because the characteristic binding energies ofthe 1s electrons of identical atoms are dependent on their

a)Author to whom correspondence should be addressed. Electronic mail:[email protected]

bonding sites and their interaction with solvent H2O molecules,it should be possible to specify the primary site of energy depo-sition by using X-rays for a particular resonant excitation of the1s electron to an unoccupied orbital. Detailed measurementsof the X-ray absorption near-edge structure (XANES) of DNAand nucleotides should therefore allow precise examinationof the primary deposition of radiation energy into a specificatomic site in the nucleobase moiety.

In related previous research,7,8 we measured the XANESspectra at energies around the N K-edge of nucleotides contain-ing purine derivatives (hereinafter referred to as purine nucle-otides), namely, adenosine 5′-monophosphate (AMP), adeno-sine 5′-triphosphate (ATP), and guanosine 5′-monophosphate(GMP) in aqueous solution. We studied nucleotides insteadof bare nucleobases to examine the direct interaction be-tween radiation and the nucleobases moiety because barenucleobases are insufficiently water soluble to allow XANESmeasurements on aqueous samples. The XANES spectra weremeasured at various pH conditions to examine the interactionof the nucleobase moiety with the solvent, and the resonantexcitation features in the energy region below the N K-edge(referred to as the pre-edge region) in the XANES spectrawere shown to be strongly dependent on the pH conditions.Two characteristic XANES peaks were identified as the core-electron transitions of N atoms with either imine (==N—) oramine (>N—) chemical bonding character. These two typesof N atoms are mutually interconverted by protonation and de-protonation (hereinafter jointly referred to as (de)protonation),and this interconversion was observed as the characteristicchange in the XANES spectrum.7,8

The aim of the present study is to advance further the sys-tematic understanding of how the interaction of the nucleobase

0021-9606/2015/142(17)/175102/9/$30.00 142, 175102-1 © 2015 AIP Publishing LLC


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175102-2 Shimada et al. J. Chem. Phys. 142, 175102 (2015)

FIG. 1. Structures of the nucleobasemoieties of (1) UMP, (2) dTMP, and(3) CMP. The canonical form of eachnucleobase is shown, which is the dom-inant form in an aqueous environment.9

The structures of uracil, thymine, andcytosine are given by the substitution ofa hydrogen atom at the respective N1atom. The (de)protonation sites (N3)are indicated by arrows together withthe (de)protonation pKa values (takenfrom Ref. 10).

moiety with the solvent affects the direct radiation interaction.To this end, we measured the XANES spectra in the N K-edgeregion of three pyrimidine-containing nucleotides (hereinafterreferred to as pyrimidine nucleotides), uridine 5′-monophos-phate (UMP), 2′-deoxythymidine 5′-monophosphate (dTMP),and cytidine 5′-monophosphate (CMP) (Fig. 1), in aqueoussolutions and in films under various pH conditions. The XANESspectra showed a characteristic dependence on the pH of thesolution. This dependence is explained by the chemical shiftof the core-levels of N atoms caused by (de)protonation. Thisinterpretation is supported by the results of theoretical calcu-lations using density functional theory (DFT). Similarities anddifferences among the spectra for these pyrimidine derivativesare discussed.

EXPERIMENTAL

Commercially obtained disodium salts of the nucleotidesUMP (TCI, Tokyo, Japan and Wako, Osaka, Japan), dTMP(Sigma-Aldrich Japan, Tokyo, Japan), and CMP (Wako, Os-aka, Japan) and the nucleobases uracil, thymine, and cytosine(TCI, Tokyo, Japan) were used as received without furtherpurification. UMP disodium salt was dissolved in purified wa-ter from a Milli-Q system (Millipore, Tokyo, Japan) to prepare0.69 mol/l (pH 7.4) and 0.61 mol/l (pH 11.1) solutions forthe liquid microjet experiment. The pH values of the solutionswere adjusted with 5 mol/l NaOH and were measured using apH meter (D-51, HORIBA, Kyoto, Japan).

The experiments were performed using beamlineBL-23SU11 at the SPring-8 synchrotron radiation facility inHyogo, Japan. The experimental setup and procedure weredescribed previously.7 Briefly, an aqueous solution of UMP isintroduced into a vacuum beamline through a 20 µm diameterorifice using a liquid microjet technique6 and is intersectedby a focused beam of monochromatized soft X-rays. Theelectrons ejected from the intersection region enter the detectorvessel though a 1 mm orifice for differential pumping. Theelectrons are collected by electrostatic lenses and detected bya channel electron multiplier without energy dispersion. Theelectron yield is normalized by the photon flux obtained fromthe drain current on the post-focusing mirror at each photonenergy and is referred to as the total electron yield. The fluxof the soft X-ray is typically around 1011 photons/s with a

band pass of 0.38 eV (full width at half-maximum; FWHM)at photon energies between 395 and 415 eV. The absolutephoton energy is calibrated against two resonant absorptionpeaks of N2 molecules in the gas phase with reference energiesof 401.10 eV for one of the 1s → π∗ vibrational peaks and406.15 eV for one of the well-characterized 1s → Rydbergstate transitions.12

The preparation of a CMP solution of sufficient concen-tration for a liquid microjet experiment is difficult under lowpH conditions because of its low solubility. Therefore, driedthin films formed using dilute solutions were used instead. Inaddition, it was difficult to obtain a sufficient amount of dTMPfrom a commercial source; therefore, dTMP film samples wereprepared in a manner similar to the CMP films. A droplet(∼20 µl) of a dilute solution (∼1 mmol/l) of CMP or dTMPwas placed on a Si plate. Drying under ambient conditions atroom temperature produced a film with an area of about 5 mm2.The samples were placed under vacuum for several hoursprior to irradiation to remove residual water and entrappedair. For comparison, films of the nucleobases uracil, thymine,and cytosine were deposited by thermal evaporation of therespective powders onto a Si plate under vacuum. The thick-ness of the films was controlled to about 100 nm using a quartzoscillator deposition monitor (CRTM-6000, ULVAC, Tokyo).Total electron yields of these film samples were obtained bymeasuring the emission current from the sample as a functionof the photon energy.

CALCULATION

A DFT method was used to calculate the XANES spectrafor the canonical form of cytosine and its N3-protonatedvariant, as well as the canonical form of uracil and thymine andtheir N3-deprotonated variants (Fig. 1). We considered onlythe canonical form and not the other tautomeric forms becausethe canonical forms of the pyrimidine moieties of CMP, dTMP,and UMP are known to be dominant in aqueous solution.9 Allsix species (both neutral and (de)protonated CMP, dTMP, andUMP) were assumed to be isolated (i.e., identical to the speciesin the gas phase). The calculation procedure was basically thesame as reported previously.8 The program package StoBe-DeMon13 is used for the calculation. The geometries of themolecules were optimized for Cs symmetry and the pyrimidine


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ring was assumed to be planar. Under Cs symmetry, molecularorbitals are classified into two irreducible representations,A′ (σ-type) and A′′ (π-type). Optimizations were conductedusing the BE88 exchange functional14 and the P86 correlationfunctional.15 The triple-zeta valence plus polarization basissets by Godbout et al.16 in a (10s6p1d)/[4s3p1d] contractionscheme were used for C, N, and O atoms, and a contracted(5s1p)/[3s1p] basis set was used for hydrogen atoms.13

The oscillator strengths fE of discrete excitations of the N1s electrons are calculated within a framework of the transitionpotential method17 using the double basis set technique18 aspreviously described.8 The calculations were performed foreach nitrogen atom N1 and N3 for uracil and thymine andadditional N4′ for cytosine. The IGLO-III basis functions19

were used to describe the core-excited N atom, whereas the1s orbitals of the non-excited N atoms were approximated byeffective core potentials. The same functionals for the geom-etry optimizations were used for calculating fE. The discretedistributions of fE obtained by this procedure were convolvedwith a Gaussian function with a FWHM of 0.7 eV to take intoaccount the vibronic broadening in an approximate manner andwere summed for two (uracil and thymine) or three (cytosine)N atoms to yield the theoretical XANES spectra I(E). Theionization potentials IPN1s of the N 1s electrons were obtainedusing the ∆KS method.20

RESULTS AND DISCUSSIONS

Experimental XANES spectra and their pH dependence

The total electron yields for UMP in aqueous solution atpH 11.1 and pH 7.4 around the N K-edge region are shownin Fig. 2. The peak structures are observed in the pre-edgeregion at about 400-403 eV and are attributed to resonantexcitations of 1s electrons of N atoms in the uracil moiety to π∗

orbitals; this is discussed in detail below. The electron yields

increase at energies above 405 eV, indicating the onset of directphotoionization of the N 1s electrons. In this post-edge region,both yield curves show two broad peaks with local maximalocated around 406 eV and 414 eV. Similar structures wereobserved in the spectrum for pyrimidine in the gas phase andwere assigned to resonant excitations to σ∗ orbitals.21

The fractional contribution to the total electron yields ofthe constituent atoms was estimated22 using the atomic crosssections23 weighted by the known chemical compositions andconcentrations of the solutions. These partial contributions arepresented in Fig. 2 in the form of stacked areas. The contribu-tions from H atoms are negligible compared with the contri-butions from the other atoms. The theoretical N 1s ionizationthresholds of uracil in the gas phase obtained from the DFTcalculations are represented by the step at 406 eV in the Natom cross sections. The experimentally obtained ratio of theyields from N atoms to those from other atoms showed goodagreement with the yields estimated from the atomic crosssections. The background contributions from atomic cross sec-tions thus estimated were subtracted from the total electronyields to obtain the XANES spectra for UMP in solution at theN K-edge, as shown in Fig. 3 by curves (1) and (3).

A different procedure for the background subtraction wasused for the film samples. The total yield in the energy rangefrom 391 eV to 397 eV below the lowest resonant peak waslinearly extrapolated to the higher energy region; this regionwas regarded as background and subtracted from the totalyield.

The XANES spectra of various pyrimidine nucleotidesin aqueous solution and in film, as well as of bare nucle-obases, are presented in Fig. 3. As described above, all theseXANES spectra exhibited isolated or overlapping peaks inthe pre-edge region and much broader maxima in the post-edge region. The XANES spectrum (3) obtained for UMP inaqueous solution at pH 7.4 (referred to as “neutral solution”below) shows two nearly overlapping resonant peaks in thepre-edge region, which are labeled E and F. The features in

FIG. 2. Total electron yields for UMP in aqueous solution at (1) pH = 11.1 and (2) pH = 7.4. Atomic cross sections weighted with the chemical composition ofthe solutions are also shown in a stacked layer representation (see text).


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FIG. 3. Experimental N K-edge XANES spectra for pyrimidine nucleotides under different acid/base conditions. N3-deprotonated UMP in aqueous solution(1) and in film (2); neutral UMP in aqueous solution (3) and in film (4); N3-deprotonated (5) and neutral (6) dTMP in film; neutral CMP in aqueous solution (7)(Ref. 6) and in film (8); and N3-protonated CMP in film (9) (see text). The spectra for the corresponding nucleobases uracil (i), thymine (ii), and cytosine (iii)are also shown.

the XANES spectrum (4) for UMP in a dried film preparedusing the same aqueous solution are essentially identical tospectrum (3). On the other hand, the XANES spectrum (1) forUMP in basic solution (pH 11.1) is strikingly different fromspectra (3) and (4) and shows four clear split resonant peaksin the pre-edge region, which are labeled A, B, C, and D. TheXANES spectrum (2) for UMP in a film prepared using thesame basic solution shows close similarity to that of (1). Inlight of previous studies on purine nucleotides,7,8 the observeddifferences in the present spectra for UMP in neutral andbasic solutions are attributed to a structural difference arounda particular N atom in the uracil moiety. The equilibriumconstant for deprotonation at the N3 site of UMP is reported tobe pKa = 9.45.10 Consequently, the N3 atom of UMP in pH 7.4aqueous solution adopts the structure shown in Fig. 3, whereasthe N3 atom is likely deprotonated in aqueous UMP at pH11.1. Thus, spectra (3) and (4) may correspond to the excitationof the N 1s electrons in the uracil moiety of the canonicalform of UMP, while spectra (1) and (2) correspond to thoseof the N3-deprotonated form. The similarity between spectra(3) and (4) of neutral UMP and spectrum (i) for uracil providesfurther support that spectra (3) and (4) correspond to the neutraluracil moiety of the canonical form. Finally, the similarity inthe XANES spectra for UMP in the aqueous solutions andtheir dried films suggests that the local environment aroundthe N3 site of UMP in the dried film is similar to that inaqueous solution, as has been shown7,8 for AMP, ATP, andGMP.

The XANES spectrum (6) in the center panel of Fig. 3was obtained using a dried film of dTMP prepared from anapproximately neutral pH aqueous solution of dTMP. Spec-trum (5) is from a dried film prepared from the dTMP solu-tion adjusted to basic pH by the addition of NaOH to thesolution used to obtain (6), although the precise pH value isnot known. Similar to UMP, a characteristic spectral changewas observed in the XANES spectra for dTMP at the two pHconditions. XANES spectrum (6) in the pre-edge region fordTMP film prepared from neutral solution shows two overlap-ping peaks, while that of spectrum (5) for dTMP film preparedfrom basic solution shows four distinguishable peaks. ThispH-dependent spectral change is similar to the one observedfor UMP. As seen in Fig. 1, the structure of the nucleobasemoiety of dTMP and UMP is essentially the same except forthe presence of a methyl group bound to the C5 atom in dTMP.Consequently, the electronic properties of the thymine moietyin dTMP can be understood similarly to those of uracil inUMP. Moreover, the acid/base properties of dTMP are similarto that of UMP, although the N3 atom of the thymine moi-ety in dTMP is deprotonated at a higher pH (pKa = 9.90).10

Therefore, the similarities in the pH dependence of the XANESspectra between dTMP and UMP indicate that this spectralchange is caused by a structural change at the N3 atom in thethymine moiety due to deprotonation. Furthermore, the pre-edge structure of spectrum (6) for dTMP in neutral solutionis similar to that of the corresponding nucleobase, thymine(ii).


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The right panel of Fig. 3 compares the spectrum (8) ofCMP in a film obtained from neutral solution to CMP inaqueous solution (7) reported previously.6 Both spectra showfour resonant peaks in the pre-edge region, labeled G, H, I,and J. The energies of these peaks in spectrum (7) for aqueoussolution are almost identical to those for film (8). However,the corresponding spectrum for cytosine film (iii) is somewhatdifferent from those for neutral CMP, as shown in (7) and(8); this is discussed below. Spectrum (9) is of a CMP filmprepared using an acidic solution (pH 2.2). Since the pH valueof this solution is below pKa = 4.33 for N3 protonation,10 thisspectrum is for the N3-protonated form of CMP. In XANESspectrum (9), three unresolved broader peaks (K, L, and M) areobserved which seem to differ from the peak profiles presentedin the XANES spectra for both cytosine (iii) and neutral CMP(7) and (8).

The data shown in Fig. 3 demonstrate that an increase inthe pH of a pyrimidine solution, i.e., from neutral UMP/dTMPto deprotonated UMP/dTMP, and from protonated CMP toneutral CMP, results in the appearance of new peaks in thelower energy region (398-400 eV). These peaks originatefrom (de)protonation at the N3 site within the pyrimidinemoiety and the consequent change in chemical shift of 1selectrons in N3. The observed systematic similarities betweenneutral UMP/dTMP and protonated CMP in the lower row of

panels in Fig. 3 and between deprotonated UMP/dTMP andneutral CMP in the upper row of panels in Fig. 3 were exam-ined further. Specifically, these experimental spectra werecompared with theoretical (calculated) spectra to clarify howstructural changes in the pyrimidine moieties induce the N K-edge XANES spectral changes.

Comparison with theoretical spectra: UMP and dTMP

The experimental XANES spectra for pyrimidine nucle-otides in aqueous solution and in film were compared withthe theoretical spectrum of the corresponding nucleobase inthe gas phase. Our purpose was to understand the XANESspectral change in terms of the chemical bonding characterof the N atoms in the nucleobase moieties in the neutral and(de)protonated forms and to confirm the above assignment ofthe spectral changes to structural changes in the pyrimidinemoieties. To our knowledge, the XANES spectra I(E) andoscillator strength fE for only the neutral forms of these pyrim-idine nucleobases and nucleotides have been reported.24,25

Fig. 4 compares the experimental XANES spectra forUMP and dTMP with the theoretical spectra I(E) for uraciland thymine in the gas phase, respectively. The upper panelsshow the spectra of the N3-deprotonated species and the lowerpanels show the spectra of the neutral species. In the present

FIG. 4. Comparison of the experimental XANES spectra for UMP in aqueous solution and dTMP in film with theoretical spectra for uracil and thymine in thegas phase. The upper panels show the spectra for N3-deprotonated species and the lower panels show the spectra for neutral species. In each panel, the upperspectrum represents the experimental spectrum for the nucleotide and the central spectrum represents the theoretical spectrum of that nucleobase. The lower partof each panel shows the theoretical oscillator strengths fE of the 1s electrons of the two N atoms plotted as vertical bars. Filled squares correspond to transitionsto π∗ orbitals and open triangles to σ∗ orbitals.


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calculation, the ionization potentials IPN1s of the N 1s electronsin N3-deprotonated uracil and thymine anions obtained usingthe ∆KS method were about 6 eV lower than those for neutraluracil and thymine. Hence, the energy region around 400 eVfor the N3-deprotonated species is embedded in the ionizationcontinuum for the N 1s electrons so that the calculated fE arequasi-continuously distributed. Similar reductions in IPN1s andoverlapping of the ionization continuum with the π∗ transitionshave been observed for the theoretical XANES spectrum of theN1-deprotonated guanine anion.8

The theoretical spectra I(E) are in good overall agree-ment with the experimental spectra. As was pointed out forthe experimental spectra, there are no significant differencesbetween the theoretical spectra for neutral uracil and neutralthymine or between their N3-deprotonated species. This is aspredicted, since uracil and thymine differ only in thymine hav-ing a methyl group at the 5′ position, which does not directlyparticipate in π-conjugation in the pyrimidine ring.

In contrast, the absence of a proton at the N3 positionalters the spectra dramatically. To investigate how the pro-ton at the N3 position affects the spectrum, we plotted thetheoretical oscillator strengths fE of the N1 and N3 1s elec-trons in the lower part of each panel in Fig. 4 as verticalbars. The filled squares represent the transition energies andfE’s of the 1s electrons to π∗ orbitals and the open trian-gles the transition energies and fE’s of the 1s → σ∗ orbitals.The lowest transition energies of N1 1s electrons to the π∗

orbital are calculated to be approximately 401 eV for uracil andthymine in both their neutral and deprotonated forms, whereasthe energies of N3 1s → π∗ are calculated to be different forthe neutral and deprotonated forms. Specifically, the ener-gies of N3 1s → π∗ for the neutral nucleobases are almostthe same as those of N1 1s → π∗, whereas the energies forthe deprotonated nucleobases are red-shifted by about 2 eV.From these plots, it is apparent that the low energy peaks Aand B in the spectra for the N3-deprotonated species are dueto transitions of the 1s electrons of deprotonated N3 to π∗

orbitals.As reported previously,7,8 the N K-edge XANES spectra

for the purine-containing nucleotides AMP, ATP, and GMPshow a systematic change in the relative intensities of theresonant peaks in the pre-edge region upon (de)protonation atthe N atoms in the purine moiety. This is ascribed to the specificchange in the 1s binding energies of the N atoms, dependingon their chemical bonding character.25–27 The 1s electrons ofimine-type (—N==) N atoms are weakly bound, whereas thoseof amine type (—N


FIG. 6. Comparison of the experimental XANES spectra for CMP with thetheoretical spectra for cytosine. The upper panel shows the spectra of neutralspecies and the lower panel the spectra of N3-protonated species. Theoreticaloscillator strengths fE of the 1s electrons of the three N atoms are also plotted.Symbols are the same as those in Fig. 4.

The spectra for both N3-protonated CMP and cytosineare shown in the lower panel of Fig. 6. Upon protonation, thecationic imine-type (—N+==) N3 atom can also take on thecharacter of an amine-type (—N


FIG. 8. Schematic electronic states of various pyrimidine nucleobases rele-vant to the present XANES spectra.

behavior of transition energies across the different pyrimidinenucleobases suggests that the final states, πL∗ and πL+1∗, of thetransitions are essentially determined by the pyrimidine ringstructure itself, and the effects of the substituents (i.e., differ-ences across uracil, thymine, and cytosine) are small. Theelectronic states of the pyrimidine nucleobases relevant to thepresent XANES spectra are depicted in Fig. 8 qualitatively.

To summarize, the XANES spectra for various species ofpyrimidine nucleotides shown in Fig. 3 are mainly character-ized according to the number {ni,na} of imine and amine Natoms. The appearance/disappearance of resonant peaks in thelower energy region (


carried out under approval of the Japan Synchrotron RadiationResearch Institute (JASRI) Proposal Review Committee, Pro-posal Nos. 2012B3810 and 2013A3810.

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日本原子力研究開発機構機関リポジトリ - JAEAand UMP are known to be dominant in aqueous solution.9 All six species (both neutral and (de)protonated CMP, dTMP, and